1. Technical Field
The present invention relates, in general, to an enzyme that degrades oxalic acid. In particular, the invention relates to the enzyme oxalate decarboxylase and to a DNA sequence encoding same. The invention further relates to a recombinant molecule comprising the oxalate decarboxylase encoding sequence and to a host cell transformed therewith. In addition, the invention relates to a method of protecting a plant from the deleterious effects of oxalic acid and to a method of reducing the oxalic acid content of a plant.
2. Background Information
Much of the oxalate from animals, including humans, originates from the oxalate ingested with plant material. Some green leafy vegetables (e.g. Amaranthus, spinach, rhubarb) are rich sources of vitamins and minerals, but they contain oxalic acid as a nutritional stress factor. Such plants, when consumed in large amounts, become toxic to humans because oxalate chelates calcium, and precipitation of calcium oxalate in the kidney leads to hyperoxaluria and destruction of renal tissues (Decastro, J. Pharm. Biomed. Anal. 6:1 (1988); Hodgkinson, Clin. Chem. 16:547 (1970)). Apart from this, at least two other instances can be cited where oxalic acid is involved in an indirect manner. In one case, the production of oxalic acid is an important attacking mechanism utilized by Whetziinia sclerotiorium, a fungus that causes serious damage to crops like sunflower. Oxalic acid accumulates in the infected tissues early in pathogenesis, and its concentration increases during the time the pathogen is colonizing the host tissues. The accumulation of oxalic acid in leaves causes symptoms of wilting and eventually leaf death. Thus, oxalic acid functions as a mobile toxin that moves from the base of stems of xylem sap and leaves (Maxwell, Physiol. Plant Pathol. 3:279 (1973)).
In another case, consumption of Lathyrus sativus (chickling vetch) causes neurolathyrism, which is characterized by spasticity of leg muscles, lower limb paralysis, convulsions, and death. L. sativus is a protein-rich hardy legume that grows under extreme conditions such as draught and water-logging and does not require complex management practices. The neurotoxin .beta.-N-oxalyl-L-.alpha.,.beta.-diaminopropionic acid (ODAP) is present in various parts of the plant. ODAP synthesis is a two-step reaction in which oxalic acid is an essential starting substrate. ODAP acts as a metabolic antagonist of glutamic acid which is involved in transmission of nerve impulses in the brain. Hence, despite its rich protein content, the legume cannot be used as a food source (Mickelson et al, (1973) in Modern Nutrition in Health and Disease: Dietotherapy (Goodhart, R. S. and Shils, M. E., Eds) 5th Ed. pp. 412-433, Lea and Febiger, Philadelphia).
A study of the function of oxalic acid in the above-mentioned systems highlights its role as an important stress factor. The value of an isolated gene encoding a protein product that degrades oxalic acid is clear. Such a gene could be used as a tool to effect degradation of oxalic acid in plants where it accumulates as such or is a substrate in the synthesis of neutoxin or is a medium for pathogenesis. This could be achieved by effecting single gene transfer to these plants.
Of the known oxalic acid-degrading enzyme systems, oxalate decarboxylase from the basidiomycetous fungus Collybia velutipes is of particular interest because of a report using partially purified enzyme that showed a simple single step breakdown of oxalic acid to carbon dioxide and formic acid, a nontoxic organic acid, in the absence of any cofactor requirement (Shimazono et al, J. Biol. Chem. 227:151 (1957)). The present invention provides purified oxalate decarboxylase and a DNA sequence encoding same. The invention also provides methods of using the encoding sequence to produce transgenic plants with low oxalic acid content. In so doing, the present invention makes possible the alleviation of stress conditions generated by oxalic acid in the cases mentioned above. It also makes possible the development of assay systems for monitoring urinary and serum oxalate levels.